An efficient preparation method of triptorelin acetate

Abstract

The present invention belongs to the technical field of pharmaceutical synthesis, and particularly relates to a method for efficiently preparing triptorelin acetate. The present invention adopts a fragment condensation method combining solid phase and liquid phase, prepares fragments 1-7 by solid phase method, prepares fragments 8-10 by liquid phase method, then condenses fragments 1-7 and fragments 8-10 by solid phase method to obtain triptorelin 10-peptide resin, and finally cleaves the resin and purifies it to obtain triptorelin acetate. This method can effectively avoid the racemization of His and Pyr, significantly reduce the D-His2-triptorelin impurity, and obtain triptorelin acetate with a purity of more than 99.9% at a total yield of up to 65%.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and particularly relates to an efficient preparation method of triptorelin acetate. Background Art

[0002] Releasing hormone drugs are a class of synthetic polypeptide drugs based on the structure of gonadotropin-releasing hormone (GnRH), and are used to treat sex hormone-dependent diseases (such as prostate cancer, endometriosis, etc.). The three commonly used releasing hormone drugs in China, namely leuprorelin, goserelin, and triptorelin, all have a huge application market.

[0003] Triptorelin, a synthetic decapeptide (containing one unnatural amino acid), is an analogue of natural gonadotropin-releasing hormone, with the molecular formula C 64 H 82 N 18 O 13 (with a molecular weight of 1311.45), and its English name is Triptorelin. Triptorelin is generally applied clinically in the form of its acetate, and is used to treat patients who need to reduce steroid hormones such as male hormones and female hormones to low levels, and is applicable to diseases such as prostate cancer, precocious puberty, endometriosis inside and outside the genital organs, female infertility, uterine fibroids, etc. The peptide sequence in the structure of triptorelin is Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 , and the structural formula is as follows:

[0004]

[0005] Patent US4010125 discloses a synthesis method of triptorelin, which uses Benzhydryl amine resin as the starting material and Boc-protected amino acids as monomers to prepare decapeptide triptorelin by stepwise condensation; patents CN200710044419.7, CN201310013712.2, and CN201310014882.2 all use Rink Amide MBHA resin or Rink Amide AM resin as the starting material and Fmoc-protected amino acids as monomers, and sequentially connect amino acids one by one to obtain the crude triptorelin after cleavage. In the above stepwise condensation method, due to the presence of Arg-Pro in the triptorelin sequence, an excessive amount of amino acids is required for solid-phase condensation under conventional conditions, and the condensation efficiency is low, and defective peptides are likely to appear. Moreover, the inventors found that there are problems of easy racemization of His and Pyr during the synthesis of triptorelin acetate by the solid-phase method, and the generation of D-His2-triptorelin impurities seriously affects the subsequent preparation and purification, resulting in problems such as low purification yield.

[0006] Patents CN201410743707.1 and CN202011087408.9 both adopt the method of solid-liquid combined fragment condensation. The former first prepares fragment one by solid phase: Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-OH; and prepares fragment two by liquid phase: Fmoc-Arg(Pbf)-Pro-Gly-NH 2 ; Then, fragment one and fragment two are condensed by liquid phase, followed by cleavage and purification to obtain pure triptorelin, with an overall yield of 38.7%. The latter first synthesizes fragment one by solid phase: Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-OH; and prepares fragment two by liquid phase: Arg(Pbf)-Pro-Gly-NH 2 ; Then, fragment one and fragment two are condensed by liquid phase, and crude triptorelin is obtained by cleavage. However, in the above-mentioned method of solid-liquid combined fragment condensation, His and Pyr are still condensed by solid phase method, and there are still the above-mentioned problems of easy racemization of His and Pyr and the generation of D-His2-triptorelin impurities, resulting in difficult purification and low yield.

[0007] Patent CN202111007822.9 discloses a method for synthesizing triptorelin in all liquid phase. Using the fragment Fmoc-Trp(Boc)-Ser(tBu)-OSu as the raw material directly, crude triptorelin is obtained after 11 steps in total. The operation is cumbersome, the raw materials are more expensive and the overall yield is low.

[0008] Based on the problems existing in the above-mentioned prior art, it is necessary to develop a new method for preparing triptorelin, which can effectively reduce the generation of impurities, improve the yield and purity of the product, and adapt to industrial production. Summary of the Invention

[0009] Based on the problems existing in the prior art, the present invention provides an efficient method for preparing triptorelin acetate. By adopting the solid-liquid combined fragment condensation method, the generation of impurities can be significantly reduced, and the target product can be obtained with a high yield.

[0010] To solve the above problems, the present invention provides the following technical solutions:

[0011] An efficient method for preparing triptorelin acetate, adopting the solid-liquid combined fragment condensation method. Fragments 1-7 are prepared by solid phase method, fragments 8-10 are prepared by liquid phase method, and fragments 1-7 and fragments 8-10 are condensed by solid phase method to obtain triptorelin 10-peptide resin.

[0012] Among them, the sequences of fragments 1-7 are the 1st to 7th amino acids of the main chain peptide sequence of triptorelin starting from the Gly end; the sequences of fragments 8-10 are the peptide chains composed of the 8th to 10th amino acids of the main chain peptide sequence of triptorelin starting from the Gly end.

[0013] Preferably, the high-efficiency preparation method of the triptorelin acetate includes the following steps:

[0014] (1) Preparation of fragments 1-7 by solid-phase method: Using Rink Amide MBHA resin as the raw material, swelling the Rink Amide MBHA resin and removing the Fmoc protection, and sequentially coupling the amino acids with N-terminal Fmoc protection and side-chain protection under the action of a coupling reagent and an activator. The order of the coupled amino acid monomers is: Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, to synthesize the fully protected triptorelin 7-peptide resin Rink Amide MBHA-Gly-Pro-Arg(pbf)-Leu-D-Trp(Boc)-Tyr(tBu)-Ser(tBu)-Fmoc;

[0015] (2) Preparation of fragments 8-10 by liquid-phase method: First, protect the N-terminal and C-terminal of H-Pyr-OH to obtain Boc-Pyr-COOR 1 , and then Boc-Pyr-COOR 1 reacts with H-His(Trt)-OH to obtain Boc-Pyr-His(Trt)-OH, and protect the C-segment of Boc-Pyr-His(Trt)-OH to obtain Boc-Pyr-His(Trt)-COOR 2 , Boc-Pyr-His(Trt)-COOR 2 reacts with H-Trp(Boc)-OH to obtain Boc-Pyr-His(Trt)-Trp(Boc)-OH;

[0016] (3) Coupling of fragments 1-7 and fragments 8-10 by solid-phase method: Remove the Fmoc protection of fragments 1-7, and then couple them with Boc-Pyr-His(Trt)-Trp(Boc)-OH prepared in step 2 under the action of a coupling reagent and an activator to obtain RinkAmide MBHA-Gly-Pro-Arg(pbf)-Leu-D-Trp(Boc)-Tyr(tBu)-Ser(tBu)-Trp(Boc)-His(Trt)-Pyr-Boc;

[0017] (4) Cleavage: Remove the peptide from the resin and simultaneously remove the side-chain protecting groups to obtain crude triptorelin peptide: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 ;

[0018] (5) The crude triptorelin peptide is purified initially and then purified by salt exchange to obtain pure triptorelin acetate.

[0019] Preferably, in step (1), during the preparation of fragments 1-7, the amino de-Fmoc protecting reagent used is a 20% piperidine DMF solution; the coupling reagent is one or a combination of Oxyma, HOBT, and HOAT; the activator is DIC; the molar ratio of Rink Amide MBHA:Fmoc-Gly-OH:coupling reagent:activator in step (1) is 1:2.4-3.6:2.4-3.6:3-3.6 (the molar ratio for subsequent coupling reactions is the same, e.g., Rink Amide MBHA-Gly-Fmoc:Fmoc-Pro-OH:coupling reagent:activator = 1:2.4-3.6:2.4-3.6:3-3.6); preferably 1:3:3.6:3.6.

[0020] Preferably, in step (2), the N-terminal protection of the amino acid is Boc protection, and the reaction condition is to react with Boc anhydride under the action of sodium carbonate. The molar ratio of amino acid:sodium carbonate:Boc anhydride is 1:1.2-2.5:1.1-1.8; preferably 1:1.5:1.2.

[0021] Preferably, in step (2), the C-terminal protecting groups R 1 and R 2 are each independently selected from one of Su and PNP, and they can be the same or different; when R 1 or R 2 is Su, the reaction condition is to react with NHS using DCC as the activator. The molar ratio of amino acid:NHS:DCC is 1:1.05-1.65:1.2-2.5, preferably 1:1.2:1.5; when R 1 or R 2 is PNP, the reaction condition is to react with PNP using DCC as the activator. The molar ratio of amino acid:PNP:DCC is 1:1.05-1.65:1.2-2.5, preferably 1:1.2:1.5.

[0022] In one embodiment, when condensing the amino acids in step (2), adding a carboxyl activator has a better effect; the carboxyl activator is sodium bicarbonate, and the molar ratio of the amino acid with both N- and C-terminal protected (such as Boc-Pyr-COOR 1): Amino acids without protection at both N / C termini (such as H-His(Trt)-OH): carboxyl activator = 1: 1.05 - 1.5: 1.2 - 2.5, preferably 1: 1.1: 1.5;

[0023] Preferably, in step (3), the coupling reagent is selected from one or a combination of Oxyma, HOBT, and HOAT; the activator is DIC.

[0024] Preferably, in step (3), the Fmoc deprotection reagent for the amino group used is a 20% piperidine in DMF solution; the molar ratio of the feed of fragment 1 - 7, fragment 8 - 10, coupling reagent, and activator is 1: 2.4 - 3.6: 2.4 - 3.6: 3 - 3.6; preferably 1: 3: 3.6: 3.6.

[0025] Preferably, in step (4), the cleavage reagent for cleaving the peptide from the resin is a mixed solution containing TFA, DTT, TIPS, and H 2 O.

[0026] In one embodiment, the volume ratio of TFA, DTT, TIPS, and H 2 O in the cleavage reagent is 85 - 92: 3 - 7: 2 - 6: 1 - 4, preferably 90: 5: 3: 2.

[0027] Preferably, in step (5), the preliminary purification is high - performance liquid chromatography purification, the chromatographic column is a C18 column, and the mobile phase system is an aqueous solution of 0.02M ammonium dihydrogen phosphate (pH 2.5) and a 0.1% TFA / acetonitrile solution.

[0028] Preferably, in step (5), the salt - changing purification is high - performance liquid chromatography salt - changing purification, the chromatographic column is a C18 column, and the mobile phase system is a 1% acetic acid / aqueous solution and methanol.

[0029] In one embodiment, the operating steps for preparing fragments 1-7 by the solid-phase method are as follows: Weigh a certain amount of Rink Amide MBHA resin and place it in a synthesis tube. Add DMF to swell the resin, and use 20% piperidine / DMF as a deprotection reagent to remove the Fmoc protection. Weigh Fmoc-Gly-OH and a coupling reagent, dissolve them in DMF, add an activator for activation, and then add them to the above synthesis tube. Use the ninhydrin method to detect and judge the reaction end point to obtain Fmoc-Gly-resin. The molar ratios of the coupling reagent, activator, and each reaction material are as described above in the present invention. Use the same method to sequentially couple the following amino acid monomers: Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, and then the fully protected triptorelin 7-peptide resin Rink Amide MBHA-Gly-Pro-Arg(pbf)-Leu-D-Trp(Boc)-Tyr(tBu)-Ser(tBu)-Fmoc can be obtained by solid-phase synthesis.

[0030] In one embodiment, prepare Boc-Pyr-COOR 1 The operating steps are as follows: Mix H-Pyr-OH with 20% acetone water, add sodium carbonate solution and Boc anhydride, and react to obtain Boc-Pyr-OH. Dissolve Boc-Pyr-OH in an organic solvent, and react with NHS or PNP under the action of an activator DCC to obtain Boc-Pyr-COOSu or Boc-Pyr-COOPNP. The organic solvent includes but is not limited to tetrahydrofuran and 1,4-dioxane. The molar ratios of each reaction material are as described above in the present invention.

[0031] In one embodiment, the operating steps for preparing fragments 8-10 by the liquid-phase method are as follows: Dissolve H-His(Trt)-OH and sodium bicarbonate in water, and dropwise add them to the solution of Boc-Pyr-COOR 1 to obtain Boc-Pyr-His(Trt)-OH. The molar ratios of Boc-Pyr-COOSu, H-His(Trt)-OH, and sodium bicarbonate for each reaction material are as described above in the present invention. Refer to the carboxyl protection step in the preparation of Boc-Pyr-COOR 1 to prepare Boc-Pyr-His(Trt)-COOR 1 ; Refer to the amino acid coupling step in the preparation of Boc-Pyr-His(Trt)-OH, and react Boc-Pyr-His(Trt)-COOR 1Couple Boc-Pyr-His(Trt)-Trp(Boc)-OH with H-Trp(Boc)-OH under the action of sodium bicarbonate.

[0032] In one embodiment, the operation steps for coupling Fragment 1-7 and Fragment 8-10 by the solid-phase method are as follows: Place Fragment 1-7 in a synthesis tube, and use 20% piperidine / DMF as the deprotection reagent to remove the Fmoc protection; Dissolve Boc-Pyr-His(Trt)-Trp(Boc)-OH and the coupling reagent in DMF, add the activator for activation, then add them to the above synthesis tube, and use the ninhydrin method to detect and judge the end point of the reaction to obtain the triptorelin 10-peptide resin; The feeding molar ratios of the coupling reagent, the activator, and each reaction material are as described above in the present invention.

[0033] In one embodiment, the preliminary purification of the crude triptorelin peptide adopts the high-performance liquid chromatography gradient elution method. The mobile phase uses 0.02M ammonium dihydrogen phosphate aqueous solution (pH 2.5) as Mobile Phase A, and 0.1% TFA / acetonitrile as Mobile Phase B. The elution gradient is as follows:

[0034]

[0035] In one embodiment, the purification and salt conversion of triptorelin adopts the high-performance liquid chromatography gradient elution method. The mobile phase uses 1% acetic acid / aqueous solution as Mobile Phase A, and methanol as Mobile Phase B. The elution gradient is as follows:

[0036]

[0037] Advantages of the present invention:

[0038] The present invention combines the advantages of solid-phase synthesis and liquid-phase synthesis to synthesize highly pure triptorelin acetate, and obtains pure triptorelin acetate peptide with a total yield of 65%. The His and Pyr that are prone to racemization under solid-phase conditions are synthesized by liquid phase, avoiding the racemization of His and Pyr, significantly reducing the impurities of D-His2-triptorelin, facilitating preparation and purification and significantly increasing the yield. The whole process is green and efficient, the method is simple, and it is convenient for industrial application. Description of the drawings

[0039] Figure 1 Chromatogram of the crude triptorelin acetate peptide

[0040] Figure 2 Chromatogram of the pure triptorelin acetate product

[0041] Figure 3 Impurity distribution diagram of triptorelin synthesized by the solid-liquid method in Example 1

[0042] Figure 4 Impurity distribution diagram of triptorelin synthesized by the full solid-phase method in Comparative Example 1 Detailed implementation manners

[0043] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims. Any simple improvement to the present invention under the premise of the method of the present invention falls within the scope of protection of the present invention.

[0044] The meanings of some commonly used English abbreviations in the present invention are shown in Table 1:

[0045] Table 1 Meanings of Commonly Used English Abbreviations in the Present Invention

[0046] English Abbreviation Meaning English Abbreviation Meaning Fmoc 9-Fluorenylmethoxycarbonyl Leu Leucine DIC N,N′-Diisopropylcarbodiimide Gly Glycine DCC Dicyclohexylcarbodiimide Arg Arginine NHS N-Hydroxysuccinimide D-Trp D-Tryptophan PNP p-Nitrophenol Tyr Tyrosine HOBT 1-Hydroxybenzotriazole Ser Serine Oxyma Ethyl 2-cyano-2-oxoacetimidate Trp Tryptophan HOAT N-Hydroxy-7-azabenzotriazole His Histidine Su Succinimidyl Pyr Pyroglutamic acid Acm Acetamidomethyl DMF N,N′-Dimethylformamide tBu tert-Butyl Piperidine Piperidine Trt Triphenylmethyl DCM Dichloromethane Boc tert-Butyloxycarbonyl NMP N-Methylpyrrolidone Asn Asparagine TFA Trifluoroacetic acid Pro Proline DTT Dithiothreitol

[0047] Example 1

[0048] 1. Preparation of Fragments 1-7 by Solid Phase Method

[0049] (1) Resin swelling: Weigh 7.0 g of Rink Amide MBHA resin with a substitution value of 0.6 mmol / g and place it in a synthesis tube. Add DMF to swell for 1 hour, and the ratio of resin to DMF is 1 g:10 ml.

[0050] (2) Resin deprotection: Add 20% piperidine / DMF to the above synthesis tube, shake and react at room temperature for 10 minutes, drain the liquid in the synthesis tube, continue to add 20% piperidine / DMF, shake and react at room temperature for 20 minutes, and wash the resin 6 times with DMF. The ratio of resin to 20% piperidine / DMF is 1 g:10 ml, and the ratio of resin to washing DMF is 1 g:10 ml.

[0051] (3) Coupling reaction: Weigh 3.75 g of Fmoc-Gly-OH and 2.04 g of HOBT, add them to 70 ml of DMF, activate with 2.33 ml of DIC under ice-water bath, then add them to the above polypeptide synthesis tube, and react at room temperature for 2 hours. Then use the ninhydrin method to detect and judge the reaction end point. If the resin is colorless and transparent, it means the reaction is complete; if the resin shows color, it means the reaction is incomplete and needs to react for another 1 hour. This judgment standard is applicable to detecting and judging the reaction end point by the ninhydrin method in subsequent amino acid couplings. The molar ratio of feedstock is Rink Amide MBHA:Fmoc-Gly-OH:HOBT:DIC = 1:3:3.6:3.6; after the reaction of 1 peptide resin, wash it 6 times with DMF.

[0052] (4) Repeat the above deprotection and coupling reaction steps to sequentially complete the coupling of Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Ser(tBu)-OH, and synthesize the triptorelin 7-peptide resin Rink Amide MBHA-Gly-Pro-Arg(pbf)-Leu-D-Trp(Boc)-Tyr(tBu)-Ser(tBu)-Fmoc by solid-phase synthesis method.

[0053] 2. Preparation of fragments 8 - 10 by liquid-phase method

[0054] (1) Synthesis of Boc-Pyr-OH: Prepare an aqueous acetone solution of 20%. Mix H-Pyr-OH with the 20% aqueous acetone solution at a mass-to-volume ratio of 1 / 10 (g / mL). Add sodium carbonate solution and Boc anhydride (the molar ratio of H-Pyr-OH:sodium carbonate:Boc anhydride is 1:1.5:1.2), stir, and keep the solution temperature below 20°C. Stir and react for 2 - 3 hours. After the reaction, add hydrochloric acid for acidification, extract with ethyl acetate, wash with acid water, wash with brine, concentrate, and crystallize with petroleum ether to obtain the product Boc-Pyr-OH.

[0055] (2) Synthesis of Boc-Pyr-COOSu: Mix Boc-Pyr-OH with tetrahydrofuran at a mass-to-volume ratio of 1 g:10 mL, add NHS, stir at room temperature until dissolved, and then add DCC. The molar ratio of Boc-Pyr-OH to NHS and DCC is 1:1.2:1.5. Stir and react at room temperature for 5 hours, filter to remove solid insoluble substances, and obtain a tetrahydrofuran solution of Boc-Pyr-COOSu.

[0056] (3) Synthesis of Boc-Pyr-His(Trt)-OH: Dissolve H-His(Trt)-OH and sodium bicarbonate in distilled water, and drop the resulting solution into the tetrahydrofuran solution of Boc-Pyr-COOSu obtained in step (2). The feeding molar ratio of Boc-Pyr-COOSu to H-His(Trt)-OH and sodium bicarbonate is 1:1.1:1.5, and the volume ratio of distilled water to tetrahydrofuran is 2:1. Stir and react at room temperature for 4 hours. Drop the reaction solution into 10 times the amount of methyl tert-butyl ether to precipitate a white solid, separate and dry to obtain Boc-Pyr-His(Trt)-OH.

[0057] (4) Synthesis of Boc-Pyr-His(Trt)-COOPNP: Mix Boc-Pyr-His(Trt)-OH and 1,4-dioxane evenly at a mass-to-volume ratio of 1 g:10 mL. Add PNP and stir at room temperature until dissolved. Then add DCC. The molar ratio of Boc-Pyr-His(Trt)-OH to PNP and DCC is 1:1.2:1.5. Stir and react at room temperature for 5 hours, and filter to remove solid insoluble substances to obtain a 1,4-dioxane solution of Boc-Pyr-His(Trt)-COOPNP.

[0058] (5) Synthesis of Boc-Pyr-His(Trt)-Trp(Boc)-OH: Dissolve H-Trp(Boc)-OH and sodium bicarbonate in distilled water, and add the resulting solution dropwise to the 1,4-dioxane solution of Boc-Pyr-His(Trt)-COOPNP obtained in step (4). The molar ratio of Boc-Pyr-His(Trt)-COOPNP to H-Trp(Boc)-OH and sodium bicarbonate is 1:1.1:1.5. The volume ratio of distilled water to 1,4-dioxane is 2:1. Stir and react at room temperature for 4 hours. Drop the reaction solution into 10 times the amount of methyl tert-butyl ether to precipitate a white solid, separate and dry to obtain Boc-Pyr-His(Trt)-Trp(Boc)-OH.

[0059] 3. Solid-phase method to couple fragments 1-7 and fragments 8-10

[0060] (1) Deprotect the triptorelin 7-peptide resin prepared in step 1. Place the triptorelin 7-peptide resin in a synthesis tube, add 20% piperidine / DMF, and oscillate and react at room temperature for 10 minutes. Drain the liquid in the synthesis tube, continue to add 20% piperidine / DMF, and oscillate and react at room temperature for 20 minutes. Wash the resin 6 times with DMF, where the ratio of resin to 20% piperidine / DMF is 1 g:10 ml, and the ratio of resin to DMF is 1 g:10 ml.

[0061] (2) Add 4.08 g of HOBT to 70 ml of DMF to the Boc-Pyr-His(Trt)-Trp(Boc)-OH prepared in step 2. After activation with 4.65 ml of DIC in an ice-water bath, add it to the polypeptide synthesis tube in (1) above and react at room temperature for 4 hours. Then detect the reaction end point by the ninhydrin method. The molar ratio of 7-peptide resin: Boc-Pyr-His(Trt)-Trp(Boc)-OH: HOBT: DIC = 1:3:3.6:3.6; after the reaction, wash 6 times with DMF and shrink 4 times with methanol to obtain 15.32 g of triptorelin 10-peptide resin.

[0062] 4. Resin cleavage

[0063] Weigh 10 g of triptorelin decapeptide resin and add it to a 250 mL three-necked round-bottom flask. Prepare 100 mL of cleavage solution according to the volume ratio of TFA:DTT:TIPS:H 2 O = 90:5:3:2. Add the cleavage solution to the round-bottom flask and react at 5 - 15 °C for 2 hours. Filter, wash the cleaved resin 3 times with a small amount of TFA, combine the filtrates, concentrate, add the concentrated liquid to ice-cold methyl tert-butyl ether for precipitation for 30 min, centrifuge, wash with methyl tert-butyl ether by centrifugation 3 times, and dry in vacuo to obtain 5.13 g of white crude triptorelin peptide with a chromatographic purity of 88.74%.

[0064] In the supplementary example, when HOBT in the above steps 1 - 3 is changed to HOAT, there is no obvious difference in the coupling reaction result, and 5.0 g of crude triptorelin peptide is obtained; when HOBT in the above steps 1 - 3 is changed to Oxyma, the coupling reaction efficiency decreases slightly, and 4.63 g of crude triptorelin peptide is obtained.

[0065] 5. Preparation and purification of triptorelin acetate

[0066] (1) Preliminary purification process: Purification is carried out by high performance liquid chromatography. The chromatographic packing material for purification is Welch ultimate C18, 5 μm, The mobile phase system is 0.02 M ammonium dihydrogen phosphate aqueous solution (pH 2.5) - 0.1% TFA / acetonitrile solution. The flow rate of a 77 mm * 250 mm chromatographic column is 70 mL / min. Gradient elution is used, and cyclic injection purification is carried out. Load the crude product solution onto the chromatographic column, start the mobile phase elution, collect the main peak, evaporate the acetonitrile, and obtain the purified and concentrated triptorelin solution.

[0067] The elution gradient is as follows:

[0068]

[0069]

[0070] (2) Purification and salt conversion process: Take the concentrated solution of the purified intermediate of triptorelin acetate, filter it with a 0.22 μm mixed microporous filter membrane, collect the filtrate for standby, and carry out salt conversion by high performance liquid chromatography. The mobile phase system is 1% acetic acid / aqueous solution - methanol. The chromatographic packing material for purification is Welch ultimate C18, 5 μm, The flow rate of a 77 mm * 250 mm chromatographic column is 70 mL / min. Gradient elution is used, and the cyclic injection method is adopted. Load it onto the chromatographic column, start the mobile phase elution, collect the chromatogram, observe the change in absorbance, collect the main peak of salt conversion and detect the purity by analytical liquid chromatography as 99.97%, and combine the solution of the main peak of salt conversion.

[0071] Elution gradient:

[0072]

[0073] (3) Post-treatment of the salt-exchanged effluent: Under the condition of a water bath at less than 50 °C, it was concentrated under reduced pressure, and most of the methanol was evaporated with a rotary evaporator to obtain an aqueous solution of triptorelin acetate, which was freeze-dried to obtain 3.58 g of pure triptorelin acetate in the form of a white powder. The purification yield was 69.78%. The total yield of the route was 65% (calculated based on the Rink resin in step 1). The chromatographic purity of the impurity of D-His2-triptorelin detected by high-performance liquid chromatography was 0.12% (attached Figure 3 ). Acetic acid did not appear in the mass spectrometry detection of triptorelin acetate. The theoretical exact molecular weight of triptorelin was 1311.45, and the mass spectrometry result of the sample was 1311.45, which was consistent with the theoretical molecular weight.

[0074] Example 2

[0075] It was carried out according to the operation steps of Example 1, except that the molar ratio of the feed of Rink Amide MBHA:Fmoc-Gly-OH:HOBT:DIC in step 1 was changed to 1:2.4:2.4:3. 3.40 g of pure triptorelin acetate in the form of a white powder was obtained, and the detected purity was 99.96%. The total yield of the route was 61.7% (calculated based on the Rink resin in step 1). The mass spectrometry result of triptorelin acetate was consistent with the theoretical molecular weight.

[0076] Example 3

[0077] It was carried out according to the operation steps of Example 1, except that the molar ratio of the feed of H-Pyr-OH:sodium carbonate:Boc anhydride in step 2 was changed to 1:1.2:1.1; the molar ratio of Boc-Pyr-OH to NHS and DCC was changed to 1:1.05:1.2; the molar ratio of Boc-Pyr-COOSu to H-His(Trt)-OH and sodium bicarbonate was changed to 1:1.05:1.2; the molar ratio of Boc-Pyr-His(Trt)-OH to PNP and DCC was changed to 1:1.05:1.2. 3.10 g of pure triptorelin acetate in the form of a white powder was obtained, and the detected purity was 99.93%. The total yield of the route was 56.3% (calculated based on the Rink resin in step 1). The mass spectrometry result of triptorelin acetate was consistent with the theoretical molecular weight.

[0078] Example 4

[0079] The operation steps of Example 1 were followed, except that the molar ratio of H-Pyr-OH:sodium carbonate:Boc anhydride in Step 2 was changed to 1:2.5:1.8; the molar ratio of Boc-Pyr-OH to NHS and DCC was changed to 1:1.65:2.5; the molar ratio of Boc-Pyr-COOSu to H-His(Trt)-OH and sodium bicarbonate was changed to 1:1.5:2.5; and the molar ratio of Boc-Pyr-His(Trt)-OH to PNP and DCC was changed to 1:1.65:2.5. 3.31 g of white powdery triptorelin acetate pure peptide was obtained, with a detected purity of 99.97% and a total route yield of 60.1% (based on the Rink resin in Step 1). The mass spectrometry results of triptorelin acetate were consistent with the theoretical molecular weight.

[0080] Example 5

[0081] The operation steps of Example 1 were followed, except that the molar ratio in Step 3 was changed to 7-peptide resin:Boc-Pyr-His(Trt)-Trp(Boc)-OH:HOBT:DIC = 1:3.6:3.6:3.6; and the volume ratio of the cleavage reagent in Step 4 was changed to TFA:DTT:TIPS:H 2 O = 92:3:2:3. 3.23 g of white powdery triptorelin acetate pure peptide was obtained, with a detected purity of 99.95% and a total route yield of 58.6% (based on the Rink resin in Step 1). The mass spectrometry results of triptorelin acetate were consistent with the theoretical molecular weight.

[0082] Comparative Example 1

[0083] The method for solid-phase synthesis of triptorelin throughout the process: The refined product of triptorelin acetate was prepared according to the operation steps in Example 1 of the specification of Patent CN103012565B, and the chromatographic purity of D-His2-triptorelin impurity detected by high-performance liquid chromatography was 2% (attached Figure 4 ).

Claims

1. A preparation method of triptorelin acetate, characterized in that, a fragment condensation method combining solid phase and liquid phase is adopted. The fragments 1-7 are prepared by the solid phase method, the fragments 8-10 are prepared by the liquid phase method, and the fragments 1-7 and the fragments 8-10 are condensed by the solid phase method to obtain triptorelin decapeptide resin; wherein, the sequences of the fragments 1-7 are the 1st to 7th amino acids of the main chain peptide sequence of triptorelin starting from the Gly end; the sequences of the fragments 8-10 are peptide chains composed of the 8th to 10th amino acids of the main chain peptide sequence of triptorelin starting from the Gly end. The preparation method specifically includes the following steps: (1) Preparation of fragments 1-7 by solid phase method: Using Rink Amide MBHA resin as the raw material, swelling the Rink Amide MBHA resin and removing the Fmoc protection, and sequentially coupling the amino acids with N-terminal Fmoc protection and side chain protection under the action of a coupling reagent and an activator. The order of the coupled amino acid monomers is: Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, to synthesize the fully protected triptorelin heptapeptide resin Rink Amide MBHA-Gly-Pro-Arg(pbf)-Leu-D-Trp(Boc)-Tyr(tBu)-Ser(tBu)-Fmoc. (2) Preparation of fragments 8-10 by liquid phase method: First, protect the N-terminus and C-terminus of H-Pyr-OH to obtain Boc-Pyr-COOR 1 , then Boc-Pyr-COOR 1 reacts with H-His(Trt)-OH to obtain Boc-Pyr-His(Trt)-OH, and protect the C-segment of Boc-Pyr-His(Trt)-OH to get Boc-Pyr-His (Trt) -COOR 2 , Boc-Pyr-His(Trt)-COOR 2 reacts with H-Trp(Boc)-OH to obtain Boc-Pyr-His(Trt)-Trp(Boc)-OH; (3) Coupling of fragments 1-7 and fragments 8-10 by solid phase method: Removing the Fmoc protection from the fragments 1-7, and then coupling with Boc-Pyr-His(Trt)-Trp(Boc)-OH prepared in step (2) under the action of a coupling reagent and an activator to obtain Rink AmideMBHA-Gly-Pro-Arg(pbf)-Leu-D-Trp(Boc)-Tyr(tBu)-Ser(tBu)-Trp (Boc)-His(Trt)-Pyr-Boc. (4) Cleavage, removing the peptide from the resin and simultaneously removing the side chain protecting groups to obtain crude triptorelin peptide: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 ; (5) The crude triptorelin peptide is subjected to preliminary purification and purification for salt exchange to obtain the pure triptorelin acetate. Among them, the amino deprotection reagent used in the solid phase synthesis process of steps (1) and (3) is a 20% piperidine DMF solution; the coupling reagent is one or a combination of Oxyma, HOBT, and HOAT; the activator is DIC. The molar ratio of Rink Amide MBHA:Fmoc-Gly-OH:coupling reagent:activator in step (1) is 1:2.4-3.6:2.4-3.6:3-3.

6. The C-terminal protecting group R in step (2) 1 and R 2 are each independently selected from one of Su and PNP, and they can be the same or different; when R 1 or R 2 is Su, the reaction condition is using DCC as an activator to react with NHS, and the molar ratio of amino acid to NHS and DCC is 1:1.05 - 1.65:1.2 - 2.5; when R 1 or R 2 is PNP, the reaction condition is using DCC as an activator to react with PNP, and the molar ratio of amino acid to PNP and DCC is 1:1.05 - 1.65:1.2 - 2.5; The molar ratio of fragments 1-7 and fragments 8-10, coupling reagent, and activator in step (3) is 1:2.4-3.6:2.4-3.6:3-3.

6. In step (4), the cleavage reagent for excising the peptide from the resin is a mixed solution containing TFA, DTT, TIPS and H 2 O; the volume ratio of TFA, DTT, TIPS and H 2 O in the cleavage reagent is 85-92:3-7:2-6:1-4.

2. According to the preparation method described in claim 1, characterized in that, In step (1), the molar ratio of Rink Amide MBHA:Fmoc-Gly-OH:coupling reagent:activator is 1:3:3.6:3.

6.

3. According to the preparation method described in claim 1, it is characterized in that in step (2), the N-terminal protection of the amino acid is Boc protection, and the reaction condition is to react with Boc anhydride under the action of sodium carbonate. The molar ratio of amino acid:sodium carbonate:Boc anhydride is 1:1.2 - 2.5:1.1 - 1.

8.

4. According to the preparation method described in claim 1, it is characterized in that The C-terminal protecting group R in step (2) 1 and R 2 are each independently selected from one of Su and PNP, and they can be the same or different; when R 1 or R 2 is Su, the reaction conditions are using DCC as an activator to react with NHS, and the molar ratio of amino acid to NHS and DCC is 1:1.2:1.5; when R 1 or R 2 is PNP, the reaction conditions are using DCC as an activator to react with PNP, and the molar ratio of amino acid to PNP and DCC is 1:1.2:1.

5.

5. According to the preparation method described in claim 1, it is characterized in that in step (3), the molar ratio of fragment 1 - 7 and fragment 8 - 10, coupling reagent, activator is 1:3:3.6:3.

6.

6. According to the preparation method described in claim 1, it is characterized in that In step (4), the cleavage reagent for cleaving the peptide from the resin is a mixed solution containing TFA, DTT, TIPS and H 2 O; the volume ratio of TFA, DTT, TIPS and H 2 O in the cleavage reagent is 90:5:3:

2.

7. According to the preparation method described in claim 1, it is characterized in that in step (5), the preliminary purification is by high performance liquid chromatography on a C18 column, and the mobile phase system is 0.02M ammonium dihydrogen phosphate aqueous solution and 0.1% TFA / acetonitrile solution.

8. According to the preparation method described in claim 1, it is characterized in that in step (5), the salt exchange for purification is by chromatography on a C18 column, and the mobile phase system is 1% acetic acid / aqueous solution and methanol.

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